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Bombyx antennal binding protein gene, protein and preparation method of protein

A technology for binding proteins and antennae, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low protein expression and difficult purification

Inactive Publication Date: 2013-05-08
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the expression and purification of silkworm antennae-binding protein, because the expression level of the protein is very low and it is difficult to purify

Method used

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  • Bombyx antennal binding protein gene, protein and preparation method of protein
  • Bombyx antennal binding protein gene, protein and preparation method of protein
  • Bombyx antennal binding protein gene, protein and preparation method of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Amplification ABP Gene fragment

[0034] Publicly available under GenBank accession number: DQ311409 ABP The gene mRNA sequence, combined with the characteristics of other homologous proteins, predicts that the first 63 bp is the sequence encoding the signal peptide, which is unnecessary, and amplifies directly from the 64th position. The primers are designed as follows:

[0035] Upstream primer F1: 5'-CG GAATTC GATAACGTCCACCTTAC-3' (SEQ ID NO: 3, underlined as Eco RI restriction site);

[0036] Downstream primer R1: 5'-GCC CTCGAG CAGTGTGTAGTAATTT-3' (SEQ ID NO:4, underlined as xho Ⅰ enzyme cleavage site).

[0037] Among them, the downstream primer was added at position 51 after the stop codon. Since the position of the downstream stop codon did not bind firmly to the downstream primer, a downstream primer was designed at a position with high GC content that could be combined.

[0038] Using the total mRNA of silkworm chrysalis as a template, cDNA ...

Embodiment 2

[0054] Example 2 Construction of recombinant vector pET-32a-ABP

[0055] The purpose obtained by the above PCR amplification ABP restriction endonuclease Eco lI and xho Ⅰ (both purchased from Fermentas) were digested with double enzymes, and the insertion was also processed Eco lI and xho Ⅰ double-enzyme-digested pET-32a vector (this vector is a prokaryotic expression vector, there are six histidine tags upstream of the restriction site where the target fragment is added, and there is a thioredoxin behind the tag, so the expressed protein is sequentially Contains HIS tag, thioredoxin, target protein. The vector can be purchased through commercial channels, such as Novagen, etc., the vector used in this example is kept in the laboratory), and the recombinant vector was successfully constructed, named pET-32a- ABP.

[0056] ABP Gene fragment double enzyme digestion system: ABP Gene fragment 41μL, Eco lI 2μL, xho Ⅰ 2 μL, 5 μL of 10× buffer, the total volume is 5...

Embodiment 3

[0061] Example 3 Expression of recombinant protein

[0062] The recombinant vector pET-32a-ABP of Example 2 was transformed into E.coli BL21 (purchased from Fermentas Company) to obtain recombinant genetically engineered bacteria (hereinafter referred to as recombinant bacteria), which contained 50 mg / mL ampicillin (Shanghai Hengyuan biological Technology Co., Ltd.) in LB medium (purchased from Oxoid Company) at 37°C, shaken at 220 rpm until OD600≈0.5, added IPTG to a final concentration of 0.5 mM, induced at 37°C for 12 hours, and centrifuged at 12,000 rpm for 1.5 mL of the bacteria solution. Add 50 μL of 1×PBS to resuspend the pellet, then take 40 μL of the resuspension, add 10 μL of 5× loading buffer, place in a metal bath at 60°C for 10 minutes, and then centrifuge at 12,000 rpm for 5 minutes to obtain a recombinant protein sample, take 15 μL for 12% SDS-PAGE, electrophoresis results such as Figure 4 . The induced cultured empty vector pET-32a(-) and the uninduced re...

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Abstract

The invention discloses a bombyx antennal binding protein gene, a protein and a preparation method of the protein. The nucleotide sequence of the bombyx antennal binding protein gene is represented by the SEQ ID NO: 1. The preparation method of the bombyx antennal binding protein gene comprises the steps of constructing a recombinant expression vector by using the bombyx antennal binding protein gene disclosed by the invention, then introducing host bacteria to construct genetically engineered bacterium, inducing by IPTG (isopropyl-beta-d-thagalactoside) to express the protein and then purifying, so as to obtain the final protein product. The ABP gene segment disclosed by the invention excludes a signal peptide sequence, the constructed recombinant expression vector pET-32a-ABP after being subjected to inducing expression can obtain a large amount of recombinant interest proteins, thus a very important basis is provided to the subsequent researches on three-dimensional structure and biological function; and by adopting the method, the problems of low expression amount and difficult purification of natural membrane proteins are also solved.

Description

technical field [0001] The invention relates to a silkworm antennae-binding protein gene, protein and a preparation method thereof. Background technique [0002] The silkworm is one of the few beneficial insects in the order Lepidoptera. Antennal binding protein (ABP) of silkworm is a subclass of odorant binding proteins (OBPs), which plays a role in the chemical information exchange between insects and the external environment. It plays an important role in foraging, courtship and reproduction of insects. A sensitive sense of smell is indispensable for the normal survival and adaptation of insects to the environment. The insect olfactory system is a highly specific and extremely sensitive chemical monitor, which can identify as low as a few parts per million from thousands of different odors. One of some specific odor substances. Most of the odorous substances sensed by insects are fat-soluble small molecular compounds. These small molecular substances diffuse through the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/11C12N15/70C12N1/21C07K14/435C12R1/19
Inventor 张耀洲李晓瑞李霞陈剑清舒特俊
Owner TIANJIN YAOYU BIOLOGICAL TECH
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